Olympus IX81 Deconvolution System.
Fluorescence microscopy is extremely powerful tool due to its ability to show
specifically labelled structures within a biological sample. However in wide
field fluorescent microscopy the images collected always contain some in
focus and out of focus light. This means that an image of a certain structure is
always blurred by the light that is present from structures above and below the
focal plane. This results in a loss of image contrast and resolution.
This process is due to the optical properties of light and how that light
behaves as it passes through the different optical components in the microscope
system. When Light is emitted from any point in the specimen a fraction of
the light is collected by the objective and focused in the corresponding point
in the microscope image plane. However the image of the point of light does not
form a point in the image plane. Instead it produce a diffraction pattern of
concentric rings of light surrounding a central disk (see image below). This
disc is called an an Airy disc. The radius of this disk is
determined by the NA of the objective lens. Higher NA objectives have a greater resolving power.
The point spread function (PSF) is the three-dimensional
diffraction pattern of light emitted from a point source in the specimen and
transmitted to the image plane. The point spread function is a well defined
physical property and can be determined accurately for most imaging systems. Deconvolution uses the knowledge we have on this
point spread function to reverse the process and reassign the light to the
correct image plane. Deconvolution microscopy uses computer-based
algorithms to remove the out of focus blur associated with wide field
fluorescent microscopy and improve resolution.
Below: 3D image of Dicty microtubules GFP-Tubulin. Convolved and deconvolved.
(Images captured on the Olympus BX81 Deconvolution Microscope using Volocity
software.Images provided by Jason King R6)
Below Dicty mitochondria images using a mitochondrial targeted GFP.